Dual Inhibitors of HIV-1 GP-120 Interactions

ABSTRACT

Compounds, which inhibit the binding of gp120 to CD4 as well as 17b and methods for their use in inhibiting the HIV fusion process, are provided.

This patent application claims the benefit of priority to U.S.Provisional Application Ser. No. 60/644,172, filed Jan. 14, 2005 andU.S. Provisional Application Ser. No. 60/637,091, filed Dec. 16, 2004,teachings of each of which are herein incorporated by reference in theirentirety.

This invention was supported in part by funds from the U.S. government(NIH Grant No. P01 GM 056550-08/C210JC) and the U.S. may have certainrights in this invention.

BACKGROUND OF THE INVENTION

Acquired immunodeficiency syndrome (AIDS), the global epidemic diseasecaused by HIV-1, has created an urgent need for new classes of antiviralagents (UNAIDS/World Health Organization (2003) AIDS Epidemic Update(UNAIDS—World Health Organization, Geneva)). The envelope glycoproteinof HIV-1 is a trimer consisting of three gp120 exterior envelopeglycoproteins and gp41 transmembrane glycoproteins (Chan et al. Cell1997, 89, 263-273; Wyatt et al. Science 1998, 280, 1884-1888; Tan et al.Proc. Natl. Acad. Sci. USA 1997, 94, 12303-12308). Viral infection isinitiated by gp120 binding to CD4 on the host cell surface (Klatzmann etal. Nature 1984, 312, 767-768; Dalgleish et al. Nature 1984, 312,763-767). The binding of these two proteins promotes a conformationalchange in gp120 that increases its affinity with a second host cellreceptor, one of the chemokine receptors, CCR5 and CXCR4 (Trkola et al.Nature 1996, 384, 184-187; Feng et al. Science 1996, 872-877; Doranz etal. Cell 1996, 85, 1149-1158; Dragic et al. Nature 1996, 381, 667-673;Wu et al. Nature 1996, 384, 179-183). The interaction of gp120 with itsreceptors is thought to promote further conformational rearrangements inHIV-1 envelope that drive fusion of the viral and host cell membranes.Blocking of these interactions between gp120 and cell surface receptorsis an attractive goal for preventing HIV-infection.

A 12- residue peptide [RINNPWSEAMM (SEQ ID NO:1)] was discovered byphage library (Ferrer et al. J. Virol. 1999, 73, 5795-5802). Its mode ofaction showed (Biorn et al. Biochemistry 2004, 43, 1928-1938) that, thepeptide inhibited the interaction of gp120 to CD4 and 17b, an antibodythat recognizes an epitope overlapping the CCR5 binding site, withmicromolar affinity. The various mutations and truncations of thepeptide confirmed that the entire sequence with the large aromaticresidue Trp next to Pro is critical for binding.

SUMMARY OF THE INVENTION

A modified peptide with 4-phenyl, 1, 4 disubstituted 1,2,3 triazole,fabricated through click chemistry, has now been identified, whichinhibits the binding of gp120 to CD4 as well as 17b at IC₅₀ values of 22and 29 nanomolar, respectively.

Accordingly, the present invention relates to compositions comprisingthis modified peptide or mutants or fragments thereof, methods fordesigning new antagonists based upon this peptide or mutants orfragments thereof and methods for using this peptide or mutants orfragments thereof and newly designed antagonists to inhibit the HIVfusion process.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the structure of a native peptide with proline.

FIG. 1B shows the structure of X in peptide 5 of the present inventioncomprising a (2S,4S)-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylic acid) substituted proline.

FIG. 2A and 2B are line graphs from experiments measuring direct bindingof peptide 5 over immobilized YU2 gp120. FIG. 2A provides responsesensorgrams for increasing concentrations (5 nmol to 5 μmol) of peptide5 binding to the immobilized YU2 gp120. FIG. 2B provides a fit of directbinding data to a steady state 1:1 binding model. Req. was calculatedfrom 280 to 295 seconds in each concentration sensorgram and plottedagainst the concentration of the peptide. Equilibrium binding constantsfor YU2-peptide 5 interaction are KA=7.99×10⁷ M⁻¹ and KD=1.28×10⁻⁸ M.

FIGS. 3A and 3B are response sensorgrams of complete inhibition ofbinding experiments of YU2 gp120 to CD4 (FIG. 3A) and 17b (FIG. 3B) bypeptide 5. The CD4 and 17b were immobilized on a CM5 sensor chip. YU2gp120 (100 nmol) was passed over the surface in the absence or presenceof 10 nmol to 1 μmol of peptide 5.

FIG. 4 is a response sensorgram of peptide 5 competition with CD4 in thereverse orientation over immobilized YU2 gp120. CD4 (50 nM) was passedover a high-density YU2 gp120 surface in absence or presence of 10 to250 nM of peptide 5.

DETAILED DESCRIPTION OF THE INVENTION

Recent advances of Cu(I)-catalyzed Huigen 1-3 dipolar cycloaddition ofazides and terminal alkynes affords 1,4-disubstituted 1, 2, 3-triazoleswith superior regioselectivity, and almost quantitative transformationunder extremely mild conditions (Rostovtsev et al. Angew. Chem. Int. Ed.2002, 41, 2596-2599; Tornoe et al. J. Org. Chem. 2002, 67, 3057-3064).The simple and robust features of this methodology have foundapplication in drug discovery, bioconjugation and material science (Wanget al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Deiters et al. J. Am.Chem. Soc. 2003, 125, 11782-11783; Link et al. J. Am. Chem. Soc. 2003,125, 11164-11165; Speers et al. Chemistry & Biology, 2004, 11, 535-546;Fazio et al. J. Am. Chem. Soc. 2002, 124, 14397-14402; Manetsch et al.J. Am. Chem. Soc. 2004, 126, 12809-12818; Helms et al. J. Am. Chem. Soc.2004, 126, 15020-15021).

In our study of the entry inhibitor, RINNIPWSEAMM (SEQ ID NO:1), we wereinterested in replacing proline of this peptide, referred to herein aspeptide 1 (structure shown in FIG. 1A), with γ-amino proline (Amp). Weused surface plasmon resonance to verify the direct interactions ofpeptides to YU2 gp120. Surface plasmone resonance analysis showed thatpeptide 4 (RINNIAmpSEAMM; SEQ ID NO:4) with cis-γ-amino proline had noeffect on gp120. However, intermediate peptide 2 (RINNIHypSEAMM; SEQ IDNO:2) and intermediate peptide 3 (RINNIAzpSEAMM; SEQ ID NO:3), withtrans-4-hydroxyproline (Hyp) and cis-4-azidoproline (Azp), respectively,retain the binding properties. Peptide 3 showed a marginally increasedbinding effect to YU2 gp120, with equilibrium constant K_(D), 2.87micromolar. Further, peptide 5 (RINNIXSEAMM; SEQ ID NO:5; structure of Xdepicted in FIG. 1B) exhibited enhanced binding affinity to gp120 andenhanced inhibition of cell surface receptor binding, as compared to thestarting peptide (SEQ ID NO:1/peptide 1).

The equilibrium constant K_(D) for all peptides 1-5, in direct bindinganalysis over immobilized gp120, are given in Table 1.

TABLE 1 Sequences of peptide and their direct binding kinetic constantswith surface immobilized YU2 gp120 Peptide SEQ ID NO/ sequence Peptidenumber K_(D) RINNIPWSEAMM SEQ ID NO:1/Peptide 1 4.46 × 10⁻⁶ MRINNIHypWSEAMM SEQ ID NO:2/Peptide 2 23.6 × 10⁻⁶ M RINNIAzpWSEAMM SEQ IDNO:3/Peptide 3 2.81 × 10⁻⁶ M RINNIAmpWSEAMM SEQ ID NO:4/Peptide 4 —RINNIXWSEAMM SEQ ID NO:5/Peptide 5 8-13 × 10⁻⁹ M

The peptides were synthesized using Fmoc-chemistry on PAL-PEG-PS resin.The trans-4-hydroxyl group of proline in peptide 2 was converted topeptide 3 through the trans-4-mesylate, followed by azide displacement.The cis-4-azido group on proline was converted to cis-4-amine usingtrimethylphosphine, dioxane water mixture (Lundquist et al. Org. Lett.2002, 4, 3219-3221). The [3+2] cycloaddition reaction was carried out onresin. The resin was suspended in acetonitrile, water, DIEA and pyridine(4:4:2:1) mixture. The phenylacetylene was added followed by a catalyticamount CuI. The peptide was cleaved from the resin using TFA. Thepeptides were synthesized individually for large quantities.

Additional experiments were conducted with peptide 5.

Increasing concentrations of peptide were passed over an immobilizedhigh density (5000 RU) surface. FIG. 2 shows the direct binding ofpeptide 5 YU2 gp120. Buffer injections and controls were subtracted forall reported data. The equilibrium constant K_(D) was calculated fromthe fit of direct binding to a steady state 1:1 binding model as afunction of Req. (280 to 295 seconds of each curve) vs. concentrationpeptide 5.

To assess the inhibition of binding of gp120 to CD4 and 17b, the analyteYU2 gp120 (100 nmol) in the absence or presence of peptide 5 was passedover immobilized CD4, 17b and control 2B6R Fab. The peptide 5 exhibitedno direct binding to CD4, 17b or control 2B6R.

FIG. 3 shows that increasing concentration of peptide 5 from 0 to 1micromolar leads to almost complete inhibition of binding of gp120 toboth sCD4 and 17b surfaces. The IC₅₀ values for peptide 5 inhibition ofbinding to YU2 gp120 to sCD4 and 17b were calculated by using thefraction of the initial rate (6-20 s) of YU2 gp120 binding in thepresence verses absence of peptide 5 and plotting these against the logof peptide concentration.

Using the same high density gp120 surface, we confirmed the inhibitionin the reverse orientation. FIG. 4 shows the inhibition of binding of100 nmol sCD4 to surface immobilized gp120 by increasing concentrationsof peptide 5.

Thus, as demonstrated herein, peptide 5 (FIG. 1B) comprising acis-γ-substituted proline (2S,4S)-4-(4-phenyl- 1H-1,2,3-triazol-1-yl)pyrrolidine-2-carboxylic acid) strongly inhibits the interaction ofgp120 to both CD4 and 17b with similar IC₅₀ values or 23 and 29 nmol,respectively. The results with this peptide encourage its utilization ininhibiting the HIV fusion process and as a lead tool in the drugdiscovery process. Mutants as well as fragments of peptide 5 are alsoexpected to exhibit similar properties as described herein. The strong(close to nanomolar) inhibition of binding of gp120 by peptide 5 to bothhost cell receptors is indicative of its utility as an antagonist of theHIV-1 fusion process and in designing new compounds, including, but notlimited to, mutants of peptide 5, fragments of peptide 5 and smallorganic molecule antagonists of the HIV-1 fusion process.

1. An isolated peptide consisting of SEQ ID NO:2 or SEQ ID NO:3, or amutant or fragment thereof.
 2. A composition comprising the peptide ofclaim
 1. 3. A method of inhibiting the binding of HIV to cell surfacereceptor CD4, said method comprising contacting cells infected with HIVwith the composition of claim 2.